The development of xylem dysfunction from resin leakage in pine wilt disease

Pine wilt disease, caused by the pinewood nematode (Bursaphelenchus xylophilus), induces xylem dysfunction even in the absence of water stress and leads to the death of pine trees. When Japanese black pine (Pinus thunbergii) is inoculated with the nematode, embolized regions develop around the inoculation site. Although nematodes are known to migrate through resin canals in infected trees, the mechanisms underlying widespread hydraulic dysfunction in the xylem remain poorly understood. We aimed to control the increase in xylem negative pressure using a blackout treatment to examine the accumulation of causal substances involved in embolism formation, to monitor the initiation and expansion of embolisms and to identify the key factors responsible for these processes.

The development of xylem dysfunction around the inoculation wound near the middle of the 1-year-old main stem was investigated using a combination of compact magnetic resonance imaging, cryo-scanning electron microscopy and X-ray micro-computed tomography. To facilitate the detection of causal substances, the decline in xylem negative pressure was suppressed using a blackout curtain. In addition, xylem pressure was measured with a pressure chamber to determine the threshold pressure for embolism formation.

In nematode-inoculated seedlings, embolisms occurred under a negative pressure close to atmospheric pressure (-0.5 MPa). In healthy trees, most resin canals were filled with resin, whereas in inoculated seedlings empty resin canals and leakage of resin into tracheids were observed around resin canals near the inoculation wound. In these regions, embolized tracheids were more abundant than resin-filled tracheids.

We propose that embolism formation induced by nematode infection is caused by the leakage of resin components into tracheids under tension following the destruction of living cells surrounding axial and radial resin canals. Our findings suggest that embolism formation associated with pine wilt disease is essentially unavoidable in natural conditions.